J. Org. Chem. 1988,53, 1855-1859 'H NMR 7.00-7.15 (m, 4), 6.19 (8, l ) , 4.28 (dd, 1, J = 7.5, 7.5), NMR 2.79 (t, 2, J = 8.1), 2.52 (m, 2), 2.21 (m, 2), 1.5-2.0 (m, 7); 147.6, 134.7, 134.4, 127.1, 126.4, 126.1,125.5,118.3,70.0,61.2, 35.9, 32.9 (2 C), 31.1, 28.4,26.1,25.4; IR (CHCl,) 3615,2920 cm-'.Anal. Calcd for C17H200:C, 84.96; H, 8.39. Found: C, 84.82; H, 8.32. Rearrangement of 32. Alcohol 32 (0.128 g, 0.52 mmol) was stirred with KH (0.24 g of 35% dispersion in mineral oil, 2.08 mmol) in 10 mL of THF under N2 a t 0 "C for 2 h. Water was added, and the solution was extracted with ether. The combined organic layers were dried (MgS04),filtered, and concentrated in vacuo to give 0.141 g of crude product containing mineral oil. Flash chromatography of 0.120 g (7:l hexane-EtOAc) gave 22.0 mg (20%) of 34a, followed by 83.9 mg (77%) of 34b that was 90% pure, containing chromatographically inseparable impurities. The data for 34a: 'H NMR 7.1-7.4 (m, 41,427 (br s, 1, w1/2 = lo), 2.32 (br s, 1, = 14), 2.80 (m, 3), 2.1-2.5 (m, 5), 1.2-1.9 (m, 5) [addition of D20 simplified the peak at 6 4.27 (ddd, 1, J = 2.9,6.1,6.1), decoupling at 6 3.32 removed a coupling from the peak at 6 4.27 (dd, J = 2.9,6.1)]; 13CNMR 128.5 (CH), 128.4 (CH), 126.6 (CH), 125.9 (CH), 73.3 (CH), 45.2 (CH), 37.4 (CH), 36.3 (CH,), 34.9 (CH,), 29.8 (CH,), 28.3 (CH,), 27.2 (CH,), 25.0 (CH,) (the 4 quaternary carbons were not observed); IR (CHCl,) 3580, ( e ) 213.4 (10335), 265.1 (2056), 271.8 2940 cm-'; UV (EtOH) A, (2000). The data for 34b: 'H NMR 7.0-7.3 (m, 4), 4.47 (ddd, 1, J 2 4,4,4), 3.27 (br s, 1, wl,, 2 7), 2.72 (m, 2), 1.2-2.60 (m, lo), 1.05 (m, 1) [decoupling a t 6 3.27 removed a coupling from the peak at 6 4.47 (dd, 1, J = 4,4)]; ' 9NMR 128.2 (CH), 126.1 (CH), 125.8 (CH), 125.0 (CH), 69.4 (CH), 46.6 (CH), 36.1 (CH), 33.9 (CH,), 33.6 (CH,), 29.7 (CH,), 28.4 (CH,), 27.3 (CH,), 24.3 (CH,) (the 4 quaternary carbons were not observed); IR (CHCl,) 3620,2980 cm-'; UV (EtOH) ,A, ( e ) 220 (10650), 266 (3520), 270 (3510). Isomerization of 34b t o 35. Treatment of 34b with 10 equiv of KH a t 0 "C for 2 h gave complete conversion to 35. Alcohol 35 waa recrystallized from hexane: mp 118-119 "C; 'H NMR 7.54 (d, 1, J = 7.6), 7.10-7.25 (m, 3), 464 (br s, 1, wl/,= 8), 2.75 (m, 2), 1.4-2.5 (m, 12); 13CNMR 141.1 (C), 135.5 (C), 134.4 (C), 128.0 (C), 127.4 (CH), 126.7 (CH), 126.1 (CH), 122.6 (CH), 64.6 (CH), 45.1 (CH), 35.2 (CH,), 31.8 (CH,), 31.6 (CH), 30.7 (CH,), 28.3 (CH,), 28.2 (CH,), 24.2 (CH,); IR (CHCl,) 3610, 2960 cm-'; UV (EtOH) A,, ( e ) 219 (18640), 264 (11177), 320 (180).
'v
1855
Spiro[cyclohexane-l,1'(2H )-3',4',5',6'-tetrahydropentalen]-2'-one (39). Ketone 8 (62.2 mg, 0.33 "01) was added to neat boron trifluoride etherate (0.5 mL, 4.07 mmol) a t 25 "C. The mixture was stirred for 45 min a t 25 "C. The reaction was quenched by addition of 2 mL of saturated sodium bicarbonate solution and extracted with two portions of ether. The combined ether layers were washed with brine, dried (MgSO,), and concentrated to give 55.2 mg of crude 39. Flash chromatography on silica gel (24:l hexane-EtOAc) gave 47.4 mg (76%) of pure 39: 'H NMR 2.78 (br s, 2), 2.51 (m, 2), 2.38 (m, 2), 2.14 (m, 2), 1.75 (m, 2), 1.39-1.58 (m, 8); 13C NMR 150.7, 137.9, 40.6, 32.5, 30.9, 30.6,25.5, 25.1,22.7 (the carbonyl and quaternary carbons were not observed); IR (neat) 2930, 2850, 1740, 1445 cm-'.
Acknowledgment. We thank the National Institutes of Health for financial support. Registry No. l a , 7766-48-5; l b , 113404-49-2;2a, 38267-20-8; 2b, 113404-50-5;3a, 113404-50-5;3b, 113404-52-7;312,113404-53-8; 3d, 113404-55-0;5a, 104923-37-7;5b, 113472-65-4;5c, 113472-66-5; 6a, 113404-57-2;6b, 113404-58-3;6c, 113404-59-4;8,113404-54-9; 9a, 113404-60-7; 9b, 113404-61-8; 9c, 113404-82-3; (22,4E)-ll, 113404-56-1; (22,42)-11, 113404-83-4; 12b, 113404-62-9; 1 2 ~ , 113404-63-0; 14, 113430-72-1; 15a, 113404-64-1; 15b, 97961-71-2; 1 5 ~113404-84-5; , (22,43)-17,54716-14-2;(22,42)-17,54716-13-1; (23,42)-17,54716-16-4; (2E,4E)-17,54716-17-5; 21a, 113404-69-6; 21b, 113404-72-1; 21c, 113404-86-7; 22a, 113404-73-2; 22b, 113404-75-4;2 2 ~113404-76-5; , 25, 113404-65-2;26, 113404-66-3; 27,113404-67-4; 28,113404-68-5; 29a, 113404-77-6;29b, 11340479-8; 29c, 113404-89-0;31,113404-70-9;32 (a-isomer), 113404-71-0; 32 (@-isomer),113472-67-6;34,113404-80-1;35, 113404-74-3;39, 113404-78-7; methyl crotonate, 18707-60-3; mesyl chloride, 1243-methyl-4-penten-1-yl 63-0; 3-methyl-4-penten-l-ol,51174-44-8; mesylate, 113404-81-2;methyl 1-cyclohexeneacetate, 53723-52-7; ethyl (E)-2-hexenoate, 27829-72-7; ethyl 3,3-dimethylacrylate, 638-10-8; ethyl (E)-3-methyl-2-pentenoate, 24410-84-2; ethyl (2)-3-methy1-2-pentenoate, 27805-84-1; ethyl 3-ethyl-3,8-nonadienoate, 113404-85-6; 3,4-dimethyl-2-(4-pentenyl)-2-cyclobutenone, 113404-87-8; (l-methyl-l-propenyl)bicyclo[3.2.0]heptan-6-one, 113404-88-9;ethyl 3,4-dihydronaphthalene-2-acetate, 63625-94-5.
Total Synthesis and Absolute Configuration of the Antibiotic Oligopeptide ( 4 5 ) - ( )-Anthelvencin A and Its 4R-(-) Enantiomer
+
Moses Lee, Diane M. Coulter, and J. William Lown* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T 6 G 2G2 Received August 14, 1987
The total syntheses of the two enantiomers of anthelvencin A [the naturally occurring isomer (4s)-(+)-la and its enantiomer (4R)-(-)-lb] with enantiomeric excess of 80 4% are described. The absolute configuration of natural anthelvencin A is thereby unambiguously assigned. The two enantiomers, (+)-la and (-)-lb, bind to duplex calf thymus DNA with constants of (1.46 f 0.01) X lo7 and (1.35 f 0.01) X lo7 M-', respectively, as determined by an ethidium displacement assay.
*
Anthelvencin A (1) is a naturally occurring oligopeptide isolated from the culture of Streptomyces venezue1ae.l It has antibiotic and anthelmintic activity.' Anthelvencin A (1) is a member of a modest class of oligopeptides, all of which are biologically active. This class of natural products includes kikumycin B (2),273noformycin (3),4
netropsin (4): and distamycin A (5)6b96(see Scheme I). The biological activities of 4 and 5 arise, in part, from their ability to bind to the minor groove of B-DNA at A.T-rich sequences.' The firm and site-specific binding of these sequence-reading oligopeptides and D N A is a net result
(1) Probst, G. W.; Hoehn, M. M.; Woods, B. L. Antimicrob. Agents Chemother. 1965, 789. ( 2 ) (a) Takaishi, T.; Sugawara, Y.; Suzuki, M. Tetrahedron. Lett. 1972, 1873. (b) Kikuchi, M.; Kumagai, K.; Ishida, N.; Ito, Y.; Yamaguchi, T.; Furmai. T.: Okuda, T. J. Antibiot.. Ser. A . 1965. 18, 243. (3) (a) Lee, M.; Lown, J. W. J. 'Org. Chem., in press. (b) Lee, M.; Kissinger, K.; Hartley, J. A.; Shea, R.; Dabrowiak, J. C.; Lown, J. W. J. Mol. Recognition, submitted.
(4) Diana, G. D. J. Med. Chem. 1973, 16,857. (5) (a) Julia, M.; Prgau-Joseph, N. Bull. SOC.Chim. Fr. 1967,4348. (b) Lown, J. W.; Krowicki, K. J. Org. Chem. 1985,50, 3774.
(6) (a) Arcamone, F.; Orezzi, P. G.; Barbieri, W.; Nicolella, V.; Penco, S. Gazz. Chim. Ital. 1967, 97, 1097. (b) Hahn, F. E.In Antibiotics HI.
Mechanisms of Action of Antimicrobial and Antitumor Agents; Corcoroan, J. W., Hahn, F. E., Eds.;Springer-Verlag: New York, 1975; p 79. (7) Zimmer, C.; Wiihnert, U. B o g . Biophys. Mol. Biol. 1986, 47, 31.
0022-326318811953-1855$01.50/00 1988 American Chemical Society
1856 J. Org. Chem., Vol. 53, No. 9, 1988
Lee et al.
Scheme I"
2ct(4s)-(+)-la (4R)-(-)-lb
I
\
We have recently studied the molecular recognition of chiral lexitropsins, such as (4S)-(+)- and (4R)-(-)-dihydrokikumycin B,3a and DNase I footprinting studies show that the natural (+) enantiomer binds more strongly to the sequence (A-T),of the NciI/HindIII fragment of pBR322 DNA than the (-) e n a n t i ~ m e r .We ~ ~ now report studies aimed at synthesizing chiral lexitropsins that possess three heterocyclic moieties and assessing the effects of chirality on DNA binding and, possibly, on biological activity.
Results and Discussion The synthetic route to anthelvencin A (1)is outlined in Scheme I1 and follows our recent synthesis of (+)- and (-)-dihydrokikumycin B.3 In the synthesis of anthelvencin A, a 4-aminopyrrole-2-carboxylatesynthon is needed. The required starting nitropyrrole ester 613 can be readily prepared from glycine ethyl ester hydrochloride and sodium nitromalonic aldehyde.', Only a limited amount of information is available on the condensation of p-aminopyrroles with carboxylic acid derivative^.'^ This has been due, in part, to the limited availability of p-aminopyrr01es.l~ Therefore, we have examined the condensation of (4s)(-)-2-pyrrolidone-5-carboxylic acid with the 0-aminopyrrole derived from catalytic reduction of ester 6. This condensation reaction was effected with dicyclohexylcarbodiimide (DCC) and a catalytic amount of 4-(dimethylamino)pyridine (DMAP) to afford ester 7 in 71% yield. The coupling reaction did not proceed without DMAP, and only tar was obtained. This is presumably because of the low nucleophilicity of the aminopyrrole combined with its high instability.
u
2
r 5
"Anthelvencin A (11, kikumycin B (21, norformycin (3), netropsin (4), distamycin A (5).
of specific hydrogen bonding, electrostatic attraction, and van der Waals interactions.8 The sequence specificities of 1-3 have not, as yet, been reported. The sequence-specific binding property of these natural products and DNA has stimulated much work in studying the interactions of netropsin (4)8b,gand distamycin A (5)1° with B-DNA. Our interest in molecular recognition has led to the design of lexitropsins, or information-reading oligopeptides, that can read DNA sequences. For example, the lexitropsin formylimidazolylimidazolylamidinium chloride has been shown to read the squence 5'-CCGT-3' uniquely." This is a result, inter alia, of hydrogen-bond formation between guanine 2-NH2 in the minor groove and N3 of the imidazole moiety.12 (8) (a) Zimmer, C. Prog. Nucleic Acid Res. Mol. Biol. 1975, 15, 285. (b) Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1985,82, 1376. (9) (a) Patel, D. J.; Kozlowski, S. A.; Rice, J. A.; Broka, C.; Itakura, K. R o c . Natl. Acad. Sci. U.S.A. 1981, 78, 7281. (b) Patel, D. J. Proc. Nutl. Acad. Sci. U.S.A. 1982, 79, 6424. (c) Gupta, G.; Sarma, M. H.; Sarma, R. H. J. Biomol. Struct. Dyn. 1984, I , 1457. (10) Klevit, R. E.; Wemmer, D. E.; Reid, B. R. Biochemistry 1986,25, 3296. (11) Kissinger, K.; Krowicki, K.; Lown,J. W.; Dabrowiak, J. C. Biochemistry 1987, 26, 5590.
6
8
Alkaline hydrolysis of ester 6 followed by acidification provided acid 8 in quantitative yield.13 Nitrile 9 can be readily prepared in high yield from 1-methyl-2-pyrrolecarboxylic acid.5b Catalytic reduction of the nitro substituent in 9 provided an unstable amine intermediate, which was coupled with the acid chloride of 8lS in the presence of triethylamine to afford 10 in 70% yield. Pinner reaction of 10 with HC1 in dry ethanol1' followed by treatment of the imidate ester intermediate with ammonia gave the amidine hydrochloride 11 in quantitative yield. The presence of the amidinium moiety in 11 is corroborated by the appearance of two exchangeable 'H NMR signals (two protons each) at 8.78 and 9.10 ppm and the disappearance of the infrared nitrile stretch of 10 at 2247 cm-'. The nitro group of 11 was reduced catalytically to give amine 12,which is unstable in solution. The amine 12,when allowed to react with the optically active (4s)(-)-2-amino-l-pyrroline-5-carboxylic acid ( 13a)3(84 f 4% ee) in the presence of DCC and DMAP, gave (+)-anthelvencin A (la)in 49% yield. The structure of la is con(12) Lown, J. W.; Krowicki, K.; Bhat, U. G.; Skorobogaty, A,; Ward, B.; Dabrowiak, J. C. Biochemistry 1986, 25, 7408. (13) Hale, W. J.; Hoyt,W. H. J. Am. Chem. SOC.1915,37, 2538. (14) Fanta,P. E. Organic. Syntheses; Wiley: New York, 1963; Collect. Vol. IV, p 844. (15) Murata, T.; Sugawara, T.; Ukawa, K. Chem. Pharm. Bull. 1978, 26, 3080. (16) Boatman, R. J.; Whitlock, H. W. J . Org. Chem. 1976, 41, 3050. (17) Pinner, A.; Klein, F. Chem. Ber. 1977, 10, 1889.
Total Synthesis of Anthelvencin A
firmed by the presence of a M - H - 2C1 ion at m/z 428 in t h e FAB-MS anlaysis, by the appearance of the 'H NMR NH3 signal at 10.52 ppm, and by comparison with the published IR and UV data of the natural product.' 'H NMR analysis of la in the presence of a chiral shift reagent [tris [3-[(trifluoromethyl)hydroxymethylene]-(+)-camphorato]europium(III)] in a 1:l DMSO-d,/CD&N solution and measurement of t h e resulting unequal diastereotopic amide NH5 signals at about 10 p p m provided t h e enantiomeric excess (ee) of la to be 80 f 4 % . Therefore, judging by the small differences in ee values of l a (80 f 4 % ) and t h e starting acid 13 (84 f 4%), racemization in the coupling reaction effected by DMAP is minimal, i.e., -2%.
CI-
U
(4S)-(-) -13a (4R)-(+) -13b
Comparison of t h e specific rotation of t h e synthetic material la with that of the natural antibiotic' establishes the absolute configuration of (+)-anthelvencin as 4s. The unnatural (4R)-(-)-anthelvencin (lb) was synthesized by using the procedure described above from acid (4R) - (+ ) - 13b.
AH,
tYH2
Q
14
n
H
1s
W e have also attempted t o prepare both enantiomers of N-methylanthelvencin A (15). Reduction of t h e nitro moiety in 145bfollowed by coupling with acid (4S)-(-)-13a gave 15. However, 15 is highly hygroscopic and unstable; thus a pure sample of N-methylanthelvencin A cannot be obtained. This high instability may be the reason why t h e normethyl form of 15 (Le., t h e natural product la) is selected for b y the producing microorganism.' Binding of (+)- and (-)-Anthelvewin A to Calf Thymus DNA. Both enantiomers of anthelvencin A, (+)-la and (-)-lb, bind to duplex native DNA. Their binding constants to calf thymus DNA determined by t h e ethidium binding assay which gives relative values'* are: 1.46 x lo7 M-' for la and 1.35 X lo7 M-' for lb. T h e binding constant for netropsin under these conditions is 1.86 X 10, M-'.lS The uncertainty for the binding constants is f O . O 1 X lo6 M-'. T h e DNA sequence specificity and the effects of chirality on DNA binding at individual sites determined by quantitative DNase and MPE footprinting, (18) (a) Morgan,. A. R.; Lee, J. S.;Pulleyblank, D. E.; Murray, N. L.; Evans,D. H. Nucleic Acids Res. 1979, 7, 547. (b) Le Pecq, J.-B.; Paoletti, C. J. Mol. Biol. 1967, 27, 87.
J. Org. Chem., Vol. 53, No. 9, 1988 1857 together with the biological activities of la and lb, will be reported in due course.
Experimental Section Melting points were determined on an Electrothermal melting point apparatus and are uncorrected. The IR spectra were recorded on a Nicolet 7199 F T spectrophotometer, and only the principal bands are reported. The 'H NMR spectra were recorded on Bruker WH-200 and WH-400 spectrometers. FAB (fast atom bombardment) mass spectra using glycerol as the matrix were determined on an Associate Electrical Industries (AEI) MS-9 and MS-50 focusing high-resolution mass spectrometer. Kieselgel60 (230-400 mesh) of E. Merck was used for flash chromatography, and precoated silica gel 60F-254sheets (Merck) were used for TLC, with the solvent system indicated in the procedure. TLC plates were visualized by using UV light or 2.5% phosphomolybdic acid in methanol with heating. All compounds obtained commercially were used without further purification unless otherwise stated. Ethanol and methanol were freshly distilled from magnesium turnings; tetrahydrofuran was distilled from sodium/benzophenone under an atmosphere of dry argon; ether was dried over sodium; methylene chloride was distilled from phosphorus pentoxide and stored over molecular sieves, 3A; triethylamine was treated with potassium hydroxide, then distilled from barium oxide, and stored over molecular sieves, 3A; dimethylformamide was distilled from barium oxide and stored over molecular sieves, 3A. Ethyl 4-Nitropyrrole-2-carboxylate (6).13 A warm (50 "C) solution of sodium nitromalonaldehyde (5.0 g, 35.7 mmol) and in 65% ethanol glycine ethyl ester hydrochloride (4.4 g, 29.4 "01) (18.5 mL) was treated with an aqueous solution of 20% sodium hydroxide (2.7 mL, dropwise). After the solution was stirred at 50 "C for 1 h, the reaction mixture was chilled (5 "C) and the precipitate collected. The crude product was recrystallized from methanol to give ester 6 as fine needles (2.60 g, 48% yield): mp 174-175 "C (lit.13mp 174 "C); TLC (5:45:55 methanol/hexane/ethyl acetate) Rf0.85; IR (Nujol) 3270, 3154, 1701, 1687, 1508, 1467, 1458, 1329, 1208 cm-'; 'H NMR (DMSO-d6)6 13.1 (s br, 1 H), 8.08 (d, 2.0, 1 H), 7.26 (d, 2.0, 1 H), 4.29 (4, 6.6, 2 H), 1.31 (t, 6.6, 3 H). Ethyl 4-(2-Oxopyrrolidine-5-carboxamido)pyrrole-2carboxylate (7). A solution of ester 6 (859 mg, 4.67 mmol) and 5% palladium on charcoal (253 mg) in methanol (20 mL) was hydrogenated at atmospheric pressure and room temperature, until TLC analysis indicated complete reduction of the nitro compound. The catalyst was removed by filtration. Concentration of the filtrate gave an oily product, which was coevaporated with dry methylene chloride (twice). Owing to the instability of the amine intermediate, it was used directly in the next step. The amine, (S)-2-pyrrolidone-5-carboxylic acid (920 mg, 7.13 mmol), 4-(dimethy1amino)pyridine (DMAP; 77.8 mg, 0.64 mmol), and dicyclohexylcarbodiimde (DCC; 1.44 g, 6.97 mmol) were dissolved in dry DMF (25 mL) at 0 "C. After 5 min at 0 "C, the solution was stirred under an atmosphere of argon for 17 h at room temperature. The urea was removed by filtration, and concentration of the filtrate in vacuo gave an oil, which was dissolved in a small amount of chloroform and set aside at 5 "C for 16 h. The resulting white crystalline material was collected and dried under high vacuum (about 80 "C) to afford 7 (881 mg, 71% yield): mp 198-202 "C; TLC (10% MeOH/CHCl,) R, 0.44; IR (Nujol) 3298, 1733,1662,1558,1450 cm-'; 'H NMR (DMSO-d,J 6 11.67 (s br, 1H), 10.08 (9, 1 H), 7.87 (9, 1 H), 7.26 (s, 1 H), 6.76 (5, 1 H), 4.23 (4,8.1, 2 H),4.12 (dd, 4.0, 5.0, 1 H), 2.31 (m, 1 H), 2.14 (m, 2 H), 1.96 (m, 1H),1.29 (t, 8.1,3 H); exact MS, m/z (relative intensity) for Cl2HI5N3O4265.1063 (M', 28). 4-Nitropyrrole-2-carboxylic Acid (8).13 A solution of ester 6 (2.0 g, 10.9 mmol) in 15% KOH solution (31 mL) was heated to gentle reflux for 4 h. The solution was chilled (about -20 "C) and acidified to about pH 1with concentrated HC1. The resulting pale yellow precipitate was extracted with ether (4 times). The combined ether extracts were dried (MgS04),and removal of the drying agent and solvent gave a pale yellow solid, which was dried in vacuo (80 "C) for 2 h to afford acid 8 (1.70 g, 99% yield): mp 210 "C dec (COzgas liberated) (lit.', mp 217 "C); IR (Nujol) 3640, 3200,1700,1575,1464,1377,1310 cm-'; 'H NMR (DMSO-d6)6
1858 J . Org. Chem., Vol. 53, No. 9, 1988
Lee e t al. Scheme 11"
9
10 I C
a
12
11
1% or 1%
CH3
NH2
2cr (4S)-(+)- l a a Reaction conditions: (a) Hz, Pd/C, MeOH room temperature; (b) acid chloride of 8, Et3N in THF; (c) HCl in dry EtOH, then dry NH,; (d) DCC, DMAP, DMF, room temperature, 16 h, (4S)-(-)-13a or (4R)-(+)-13b.
13.21 (s br, 1 H), 12.9 (s br, 1 H), 7.86 (d, 1.7, 1 H), 7.06 (d, 1.7, 1 H).
3-[ 1-Methyl-4-(4-nitropyrrole-2-carboxamido)pyrrole-2carboxamido]propionamidine Hydrochloride (11). A chilled 3 4 l-Methyl-4-(4-nitropyrrole-2-carboxamido)pyrrole-2- (0 "C) suspension of nitrile 10 (994.6 mg, 3.01 mmol) in dry ethanol carboxamido]propionitrile (10). A suspension of acid 8 (761 (4 mL) was bubbled with dry hydrogen chloride for 15 min. The mg, 5.59 mmol) in dry dimethoxyethane (DME) (12 mL) and dry resulting solution was stirred a t 0 OC for an additional hour and triethylamine (1.3 mL) was stirred a t ambient temperature for a t room temperature for 30 min. Removal of the solvent under reduced pressure gave a solid residue, which was washed with dry 10 min. The reaction mixture was concentrated, resuspended in dry DME (13 mL), and chilled (0 "C). Thionyl chloride (8.5 mL) ether (once). The residue was redissolved in dry EtOH (42 mL) in dry DME (8.5 mL) was then added slowly. After 15 min, the and chilled (0 "C). Dry ammonia was condensed into the reaction resulting solution was concentrated to dryness, and the acid mixture. After 50 min a t 0 "C, the solvent was removed to give chloride product was coevaporated with dry CHZC1, (twice). a pale brown solid, which was washed with carbon tetrachloride A suspension of nitrile 9 (1.01 g, 5.16 mmol) and 5% Pd on and filtered. The residue was washed with 2-propanol and CCll charcoal (360 mg) in methanol (29.0 mL) was hydrogenated a t and then dried under high vacuum a t 80 "C to give amidine 11 room temperature and atmospheric pressure to give the unstable (1.15 g, 96% yield): NMR and TLC analyses showed that 11 is amine intermediate. The oily amine was coevaporated with dry pure; mp 275 "C dec; TLC (10855 MeOH/CHCl,/HOAC) Rr 0.33; CHzClz(twice) to give a foamy material.5b The amine was susIR (Nujol) 3200 (br), 1696,1639,1580,1553,1500,1466,1370,1312 pended in dry T H F (22.0 mL) containing dry triethylamine (1.3 cm-'; 'H NMR (DMSO-d,) 6 12.88 (s br, 1H), 10.49 (s, 1 H), 9.10 mL) and chilled (-20 "C). A solution of the acid chloride prepared (s br, 2 H), 8.78 (s br, 2 H), 8.36 (t, 6.4, 1 H), 7.97 (s, 1 H), 7.67 from 8 in dry CH2C12(13 mL) was added dropwise. After 15 min (s, 1 H), 7.29 (s, 1 H), 6.94 (s, 1 H), 3.84 (s, 3 H), 3.53 (9, 6.4, 2 a t -20 "C, the reaction mixture was stirred under argon at room H), 2.67 (t, 6.4, 3 H), after DzOexchange 7.95 (s, 1 H), 7.57 (s, temperature for 16 h. The solvent was removed under reduced 1 H), 7.22 (s, 1 H), 6.85 (s, 1 H), 3.77 (s, 3 H), 3.50 (t, 6.2, 2 H), pressure to give a solid, which was washed with water (twice), 2.60 (t, 6.2, 3 H); FAB-MS, m / t (relative intensity) carbon tetrachloride (once), and hexane (once) to give a tan (C14H17N704)H+ 348, found 348 (M - C1, 31). Anal. Calcd for crystalline product 10 (1.20 g, 70% yield), which was homogeneous CI4Hl8N7O4C1 (383.45): C, 43,8 H, 4.7; N, 25.6. Found: C, 43.5; by TLC and NMR analyses: mp 255 "C dec; TLC (5:45:55 H, 4.6; N, 26.0. MeOH/hexane/ethyl acetate) R, 0.32; IR (Nujol) 3388,3270,2247, 3 4 1-Methyl-4-[4-(2-amino-1-pyrroline-5-carboxamid0)pyrrole-2-carboxamido]pyrrole-2-carboxamido]propion1637,1465,1376,1310cm-'; 'H NMR (DMSO-d,) 6 12.88 (s br, 1 H), 8.42 (t, 6.3, 1 H), 7.96 (s, 1 H), 7.61 (s, 1 H), 7.26 (s, 1 H), amidine Dihydrochloride. (4.63)-(+)-Anthelvencin A (la). A suspension of amidine 11 (250 mg, 0.65 mmol) in methanol (60 6.87 (s, 1 H), 3.83 (s, 3 H), 3.40 (4,6.3, 2 H), 2.74 (t, 6.3, 2 H); exact MS, m / z (relative intensity) for C14H14N604 330.1069 (M+, mL) was hydrogenated over 10% Pd on charcoal (125 mg) at room temperature and atmospheric pressure. The reaction was stopped 46).
Total Synthesis of Anthelvencin A when TLC analysis showed complete disappearance of the starting material 11. Removal of the catalyst and concentration of the filtrate gave a residue, which was coevaporated with dry CHzClz (twice) to give the amine intermediate 12 as a glassy green solid 'H NMR (DMSO-$) 6 10.67 (9, 1 H), 9.65 (s, 1 H), 8.97 (s br, 2 H), 8.61 (s br, 2 H), 8.21 (t, 6.0, 1H), 7.18 (s, 1 H), 6.84 (9, 1 H), 6.39 (s, 1 H), 6.27 (s, 1 H), 3.80 (9, 3 H), 3.50 (4, 6.0, 2 H), 3.20 (s br, 2 H), 2.61 (t, 6.0, 3 H), and after DzO exchange 7.13 (s, 1 H), 6.79 (9, 1H), 6.40 (5, 2 H), 3.76 (5, 3 H), 3.50 (t,6.0, 2 H), 2.60 (t, 6.0, 3 H). Owing to the instability of 12, it was used directly in the next step. The mixture of amine 12, (4S)-(-)-2-amino-l-pyrroline-5carboxylic acid hydrochloride (13a) (145.6 mg, 0.89 mmol), and DMAP (11.4 mg, 0.10 mmol) was dissolved in dry DMF (3.5 mL) and chilled (0 "C). A solution of DCC (185.5 mg, 0.90 mmol) in dry DMF (3.5 mL) was added. After 15 min at 0 "C, the solution was stirred under an atmosphere of argon at ambient temperature for 16 h. The urea was removed by filtration, and removal of the solvent under reduced pressure gave a solid residue. Water (10 mL) was added, and the solid that precipitated was removed by filtration. Concentration of the aqueous extract gave a solid residue, which was redissolved in methanol and the hot solution decolorized with activated charcoal. The filtrate was concentrated to a small volume, and acetone was added to crystallize (4s)(+)-anthelvencin A (la) as a white solid. The product was collected by filtration, and the residue was quickly dried in vacuo (80 "C), to avoid decomposition, to afford la (158 mg, 49% yield) which is homogeneous by TLC analysis: mp 170-175 "C dec; TLC (90:5:5 MeOH/CHCl,/HOAC) Rf0.17; [ a I z 4+~7.7" ( c 0.0030, HzO) [lit.' [alZzD +9.7" (H20)];UV (HzO) 238 (e 3.95), 292 nm (4.03) (lit.' 235, 300 nm); IR (Nujol) 3127 (br), 1689, 1642,1584, 1527,1405cm-' [lit.' (mineral oil) 3120 (br), 1690,1635,1580,1530, 1400 cm-'1; 'H NMR (DMSO-d,) 6 11.44 (s br, NH4), 10.52 (9, NH3), 10.09 (s, NH5), 9.04 and 9.01 (both s, 7 amidine H's), 8.37 (t, 5.8, NH7), 7.23, 7.16, 6.97, 6.89 (4 d, 1.2, H14, H9, H7, H12), 4.60 (dd, 4.9, 8.6, H4), 3.81 (s, N6-CHJ, 3.51 (q, 5.8, H17), 2.85 (t, 7.7, H2), 2.64 (t,5.8, H18), 2.07 (m, H3), and after DzO exchange 7.17, 7.13, 6.88, 6.79 (4 d, 1.6, H7, H9, H12, H14), 4.51 (dd, 4.9, 8.6, H4), 3.73 (s, N6-CH,), 3.66 (t, 5.0, H17), 2.85 (m, H2, H3), 2.64 (t,5.0, H18), 2.04 (m, H3); FAB-MS, m/z (relative intensity) (C19H25N903)H+ 428, found 428 (M - H - 2C1,2), 315 (17), 209 (8), 123 (100). The preparation of (4R)-(-)-lb followed the procedure given above. The characterization data for (-)-lb are identical with -8.0" ( c 0.0063, H2O). those of (+)-laexcept for [a]24D N-Methylanthelvencin A (15). A suspension of amidine 14
J. Org. Chem., Vol. 53, No. 9, 1988
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(200 mg, 0.54 mmol) and 5% Pd on carbon (307 mg) in methanol (25 mL) was hydrogenated at atmospheric pressure and room temperature. Removal of the catalyst and solvent gave a residue, which was coevaporated with dry CH2C12(twice) to give a foamy amine intermediate. The amine, (4S)-(-)-2-amino-l-pyrroline5-carboxylicacid hydrochloride (13a) (119.2 mg, 0.72 mmol), and DMAP (8.9 mg, 0.07 mmol) were dissolved in dry DMF (8 mL) and chilled (0 "C). A solution of DCC (167.9 mg, 0.82 mmol) in dry DMF (4 mL) was added. After 15 min at 0 "C, the ice bath was removed, and the solution was stirred at room temperature for 16 h. The urea was removed by filtration, and concentration of the filtrate gave an oily residue. The residue was washed with CCl,, and the product was extracted with water (10 mL). The resulting precipitate was removed by filtration, and removal of the solvent gave a brown oily residue, which was redissolved in MeOH and decolorized with charcoal. The filtrates was concentrated to a small volume, and acetone was added to precipitate 15 as a pale brown solid. All attempts to isolate the product by filtration failed to give any good-quality material. The product is hygroscopic and unstable in air and decomposes readily during the filtration procedure: IR (Nujol) 3120 (br), 1688, 1648, 1584, 1522, 1402 cm-1; 'H NMR (DMSO-d,) 6 10.39 (9, 1 H), 9.96 (s, 1 H), 8.38 (t, 5.5, 1 H), 7.23, 7.22, 6.92, 6.89 (4 s, 1 H each), 4.48 (dd, 4.5, 8.0, 1 H), 3.83, 3.81 (2 s, 3 H each), 3.48 (4, 5.5, 2 H), 2.80 (m, 2 H), 2.55 (t, 5.5, 3 H), 2.45 (m, 1 H), 2.00 (m, 1H), and after D20 exchange 7.14 (s, 2 H), 6.81 (s, 1H), 6.78 (s, 1 H), 4.51
(dd,4.5,8.0,1H),3.76,3.72(2~,3Heach),3.50(t,5.7,2H),2.80 (m, 2 H), 2.57 (t, 5.7, 3 H), 2.45 (m, 1 H), 2.00 (m, 1 H); FAB-MS, m / z (relative intensity) (CzoHZ7N9O3)H+ 451, found 451 (M - H - 2c1, 1).
Acknowledgment. This investigation was supported by grants (to J.W.L.) from t h e Biotechnology Strategic Grants Programme of the Natural Sciences and Engineering Research Council of Canada and by the National Cancer Institute of Canada. D.M.C. thanks t h e Alberta Heritage Foundation for Medical Research for a summer studentship. Registry No. la, 113598-28-0; lb, 113598-34-8;6,5930-92-7; 6 (4-amino deriv), 67318-12-1; 7, 113598-29-1;8, 5930-93-8; 8 (acid chloride), 28494-49-7;9,3185-95-3;9 (4-amino deriv), 97950-77-1; 10,113598-30-4;11,113598-31-5; 12,113598-32-6;13a, 110971-81-8; 13b, 110971-86-3;14, 14555-80.7; 14 (4-amino deriv), 97950-75-9; 15, 113598-33-7; H-Gly-OEt.HC1, 623-33-6;sodium nitromalonacid, 98-79-3. aldehyde, 34461-00-2;(S)-2-pyrrolidone-5-carboxylic
